Methods and compositions for modulating pd1

ABSTRACT

Disclosed herein are methods and compositions for modulating expression of a PD1 gene.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 12/927,557, filed Nov. 17, 2010, now allowed, which is acontinuation-in-part of U.S. application Ser. No. 12/284,887, filed Sep.25, 2008, which claims the benefit of U.S. Provisional Application No.60/995,566, filed Sep. 27, 2007 and U.S. Provisional Application No.61/281,432, filed Nov. 17, 2009. The disclosures of all the foregoingapplications are hereby incorporated by reference in their entireties.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Not applicable.

TECHNICAL FIELD

The present disclosure is in the fields of genome engineering andnuclease identification.

BACKGROUND

Nucleases, including zinc finger nucleases and homing endonucleases suchas SceI, that are engineered to specifically bind to target sites havebeen shown to be useful in genome engineering. For example, zinc fingernucleases (ZFNs) are proteins comprising engineered site-specific zincfingers fused to a nuclease domain. Such ZFNs have been successfullyused for genome modification in a variety of different species. See, forexample, United States Patent Publications 20030232410; 20050208489;20050026157; 20050064474; 20060188987; 20060063231; and InternationalPublication WO 07/014,275, the disclosures of which are incorporated byreference in their entireties for all purposes. These ZFNs can be usedto create a double-strand break (DSB) in a target nucleotide sequence,which increases the frequency of homologous recombination at thetargeted locus more than 1000-fold. In addition, the inaccurate repairof a site-specific DSB by non-homologous end joining (NHEJ) can alsoresult in gene disruption. Creation of two such DSBs results in deletionof arbitrarily large regions.

The programmed death receptor (PD1, also known as PDCD1) has been shownto be involved in regulating the balance between T cell activation and Tcell tolerance in response to chronic antigens. During HIV1 infection,expression of PD1 has been found to be increased in CD4+ T cells. It isthought that PD1 up-regulation is somehow tied to T cell exhaustion(defined as a progressive loss of key effector functions) when T celldysfunction is observed in the presence of chronic antigen exposure asis the case in HIV infection. PD1 up-regulation may also be associatedwith increased apoptosis in these same sets of cells during chronicviral infection (see Petrovas et al, (2009) J Immunol. 183(2):1120-32).PD1 may also play a role in tumor-specific escape from immunesurveillance. It has been demonstrated that PD1 is highly expressed intumor-specific cytotoxic T lymphocytes (CTLs) in both chronicmyelogenous leukemia (CML) and acute myelogenous leukemia (AML). PD1 isalso up-regulated in melanoma infiltrating T lymphocytes (TILS) (seeDotti (2009) Blood 114 (8): 1457-58). Tumors have been found to expressthe PD1 ligand (PDL) which, when combined with the up-regulation of PD1in CTLs, may be a contributory factor in the loss in T cellfunctionality and the inability of CTLs to mediate an effectiveanti-tumor response. Researchers have shown that in mice chronicallyinfected with lymphocytic choriomeningitis virus (LCMV), administrationof anti-PD1 antibodies blocked PD1-PDL interaction and was able torestore some T cell functionality (proliferation and cytokinesecretion), and lead to a decrease in viral load (Barber et at (2006)Nature 439(9): 682-687). Disregulation of PD1 may also play a role inautoimmune disease. SNPs of PD1 (in particular PD 1.3) have also beenassociated with increased risk for systemic lupus erythematosus (SLE).It has been shown that SLE patients have a higher frequency of the PD1.3 PD1 allele, and that these patients show reduced PD1 expression ontheir activated CD4+ T cells (see Bertsias et al, (2009) ArthritisRheum. 60(1):207-18).

Thus, there remains a need for additional PD1-targeted modulators, forexample PD1-targeted nucleases or transcription factors that can be usedin research and therapeutic applications.

SUMMARY

The present disclosure relates to development of PD1-targeted nucleases,for example engineered meganucleases and zinc finger nuclease (ZFNs).

The present disclosure demonstrates active zinc finger proteins specificfor human and rodent PD1 and fusion proteins, including zinc fingerprotein transcription factors (ZFP-TFs) or zinc finger nucleases (ZFNs),comprising these PD1-specific zinc finger proteins. The proteinscomprising PD1 specific zinc finger proteins of the invention may beused for research and therapeutic purposes, including for treatment ofany disease or disorder in which PD1 is aberrantly expressed, or wherethe PD1 pathway is aberrantly utilized due to overexpression of a PD1ligand. For example, zinc finger nuclease targeting of the PD1 locus inT cells can be used to block PD 1-dependent immune suppression in bothchronic infectious diseases and malignancies. Alternatively, a defectivePD1 locus may be remedied using ZFN dependent targeted insertion of wildtype sequences, or a zinc finger protein transcription factor (ZFP TF)may be used to modulate (e.g., upregulate or downregulate) defective PD1expression. Further, a ZFP TF targeting the PD1 locus may be used tomodulate a wild type PD1 gene.

In another aspect of the invention, the fusion proteins comprise zincfinger nucleases (ZFNs) that are specific for the human PD1 gene. Incertain embodiments, the zinc finger domains of the nuclease fusionproteins comprise the non-naturally occurring recognition helices shownin Table 1 and/or bind to the target sites shown in Table 2.

In yet another aspect, provided herein are ZFP-TFs capable of modulatingthe expression of a PD1 gene. In certain embodiments, the zinc fingerdomains of the ZFP-TFs comprise the non-naturally occurring recognitionhelices shown in Tables 1 or 5 and/or bind to the target sites shown inTables 2 or 6.

In another aspect, provided herein are methods and compositions for theregulation of the PD1 gene. In certain embodiments, the methods compriseintroducing a fusion protein comprising a zinc finger protein that isengineered to bind to a target site in a PD 1 gene (or polynucleotideencoding a fusion protein) into cells from a patient with a disease ordisorder in which the disease or disorder is characterized by aberrantexpression of PD1 and/or undesirable use of the PD1 pathway, caused byoverexpression of the PD1 ligands. The methods may be utilized in thetreatment and/or prevention of chronic infections such as HIV and HCV.Similarly, the methods and compositions may be utilized in the treatmentand/or prevention of cancer and malignant disease. Non-limiting examplesof cancers that can be treated and/or prevented include lung carcinomas,pancreatic cancers, liver cancers, bone cancers, breast cancers,colorectal cancers, leukemias, ovarian cancers, lymphomas, brain cancersand the like.

The methods and compositions described herein may be used as astand-alone treatment, or may be used in combination with otheranti-viral or anti-cancer therapies. These methods and compositions maybe provided with anti-viral or anti-cancer therapies in a sequentialfashion, or may be administered concurrently. The methods andcompositions provided may also be used to modulate PD1 expression in apatient afflicted with an autoimmune disease, or may be used to treatsuch a patient by integrating in a wild type PD1 allele, or a PD1 allelewith altered characteristics if this patient carried a defective orundesirable allele. Cell lines may be constructed to specifically alterthat PD1 gene sequence to create screening systems for therapeuticcompounds which may alter the regulation or functionality of a PD1 gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the disruption of the PD1 gene in human PBMCsusing PD1 specific zinc finger nucleases, as determined by the Cel-1based SURVEYOR™ Nuclease assay which measures the percentage ofmutations inserted by non-homologous end joining (NHEJ) that is inducedwhen the indicated ZFNs are introduced into these cells. Cells weretreated with ZFN pairs that each combined the ZFN 12942 with a differentZFN variant that bound on the opposite strand of DNA and would form afunctional nuclease upon binding to the target locus with 12942. The SBSnumber for the second ZFN of the pair is indicated below each graph. Theleft-most bar of each pair shows NHEJ percentages three-days postnucleofection for cells incubated at 37° C. The bar second from the lefton each indicated pair shows NHEJ percentages ten-days postnucleofection for cells incubated at 37° C. The bar second from theright on each indicated pair shows NHEJ percentages three-days postnucleofection for cells incubated at 30° C. and the right-most bar ofeach indicated pair shows NHEJ percentages ten-days post nucleofectionfor cells incubated at 30° C.

FIG. 2 depicts the results of an analysis to determine the sequence (SEQID NO: 81) of the PD1 locus in CD8+ T cells following treatment with thePD1 specific ZFN pair 12942 and 12947 which target exon 1. Insertionsare depicted in capitol letter in bold (SEQ ID NO 82-95). Deletions aredenoted with a (−) (SEQ ID NOS 96-110). As can be seen from the figure,several insertions and deletions were observed near the ZFN cut site asa result of DSB repair via NHEJ.

FIG. 3 depicts the results following transfection of splenocytes derivedfrom Pmel TCR transgenic/Rag1−/− mice with murine PD1-specific ZFNs.Cells were stimulated with anti-CD3 antibodies, and then stained forPD1. The plots show the percent PD1 positive CD3+CD8+ cells in eachgroup and the median PD1 fluorescence for each group, and the data isrepresented in table format below. As can be seen in the figure, PD1expression decreases in cells that received the PD1-specific ZFNs, evenin the presence of CD3 stimulation.

FIG. 4 demonstrates that the reduction in PD1 expression was evident atlater time points. Cells were also harvested at 72 hours post-CD3stimulation, and stained for PD1. The upper histograms show the percentPD1 positive CD3+CD8+ cells in each group and the median PD1fluorescence for each group. Lower plots show the frequency of PD1/CFSEexpressing cells. This figure demonstrates that PD 1 expression is stilldecreased in the cells treated with the PD1-specific ZFNs even 72 hourspost CD3 stimulation.

FIG. 5 depicts the results for the PD1 specific ZFN pairs tested in CD4+T cells and analyzed using the Cel-I assay for genome editing activity.In these cells, up to 44% editing was observed with some pairs.

FIG. 6 depicts the purification of PD1 (−) cells following treatmentwith the PD1 specific ZFN pairs into CD4+ T cells. Percent editing orNHEJ was measured by the Cel-I assay as described above, and up to 44%editing was observed with some of the PD1-specific ZFN pairs. Followingtreatment, the cells were stimulated with a first exposure toanti-CD3/CD8 beads to induce the ZFN transgenes and then re-stimulatedand subjected to a purification procedure, either by FACs or by affinitychromatography. Cells were collected and analyzed for PD1 editing by theCel-I assay (described above), i) following the first stimulation, ii)following the second stimulation but prior to any purification, iii)following cell sorting for CD25+ (a marker of activation), PD1(−), oriv) after affinity chromatography. As shown, using the cell sortingtechnique, up to 56% of the recovered cells were found to be modified.PD1(−) cells purified by the affinity chromatography displayed anoverall PD1 modification of up to 42% as assayed by Cel-1 analysis.

FIG. 7 is a graph depicting results for PD1 specific ZFNs tested in CD8+T cells. In this experiment, mRNAs encoding the PD1 specific ZFNs weretransduced into CD8+ T and the percent PD1 modification was analyzed bythe Cel I assay. The amount of modification observed was related to theamount of mRNA used, with lesser amounts of input mRNA resulting inlesser percentages of target modification. These results demonstratethat the PD1 specific ZFNs described herein are capable of modifying thePD1 locus in cell lines and in primary T cells.

DETAILED DESCRIPTION

Described herein are compositions and methods for high throughput invivo screening systems for identifying functional nucleases. Inparticular, the assays use a reporter system to monitor the ability of anuclease to induce a double-stranded break at their target site. Inaddition, the assays can be used to determine the effect of the nucleaseon cell growth (toxicity).

Engineered nuclease technology is based on the engineering of naturallyoccurring DNA-binding proteins. For example, engineering of homingendonucleases with tailored DNA-binding specificities has beendescribed. Chames et al. (2005) Nucleic Acids Res 33(20):e178; Arnouldet al. (2006) J. Mol. Biol. 355:443-458. In addition, engineering ofZFPs has also been described. See, e.g., U.S. Pat. Nos. 6,534,261;6,607,882; 6,824,978; 6,979,539; 6,933,113; 7,163,824; and 7,013,219.

In addition, ZFPs have been attached to nuclease domains to createZFNs—a functional entity that is able to recognize its intended genetarget through its engineered (ZFP) DNA binding domain and the nucleasecauses the gene to be cut near the ZFP binding site. See, e.g., Kim etal. (1996) Proc Natl Acad Sci USA 93(3):1156-1160. More recently, ZFNshave been used for genome modification in a variety of organisms. See,for example, United States Patent Publications 20030232410; 20050208489;20050026157; 20050064474; 20060188987; 20060063231; and InternationalPublication WO 07/014,275.

Although the rules that allow engineering of ZFPs to bind to specificDNA sequences are well characterized and accurately identify specificZFPs, these same ZFPs may not bind with equal affinity and/orspecificity when incorporated into a ZFN. For example, it is likely thatthe chromosomal substrate can affect the precise dimerization ofnuclease domains in living cells, consequently diminishing the cleavagepotential, and that the precise chromatin architecture over a givengenomic locus will differentially affect the ability of ZFNs to bind andcleave their intended target sequence. In addition, it is difficult ifnot impossible for in vitro assays to mimic the search parameters that adesigned DNA binding domain is subjected to when presented with acellular genome in chromatinized form. As a result, it is essential totest numerous variants in the relevant organism, or cell lineage, toidentify a ZFN displaying the optimal characteristics for genemodification.

Furthermore, since every in vivo system has its own peculiarities, it isnecessary to develop specific detection assays to determine ZFN action.Thus, unlike previously described in vivo screening methods which screenfor homing endonucleases with binding specificity different from thenaturally occurring homing endonuclease, the methods described hereinprovide a rapid and efficient way of ranking nucleases already known tobind to a particular target site by predicting their in vivofunctionality as well as the toxicity of a nuclease to the host cell.

Thus, the methods and compositions described herein provide highlyefficient and rapid methods for identifying nucleases that arebiologically active in vivo. In addition to accurately predicting invivo nucleases functionality, the assays described herein also can beused to determine nuclease toxicity, thereby allowing identification ofthe safest and most functionally active proteins.

General

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Secondedition, Cold Spring Harbor Laboratory Press, 1989 and Third edition,2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, New York, 1987 and periodic updates; the series METHODS INENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE ANDFUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolffe,eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

DEFINITIONS

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of a correspondingnaturally-occurring amino acids.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶ M⁻¹or lower. “Affinity” refers to the strength of binding: increasedbinding affinity being correlated with a lower K_(d).

A “binding protein” is a protein that is able to bind non-covalently toanother molecule. A binding protein can bind to, for example, a DNAmolecule (a DNA-binding protein), an RNA molecule (an RNA-bindingprotein) and/or a protein molecule (a protein-binding protein). In thecase of a protein-binding protein, it can bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or moremolecules of a different protein or proteins. A binding protein can havemore than one type of binding activity. For example, zinc fingerproteins have DNA-binding, RNA-binding and protein-binding activity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP.

Zinc finger binding domains can be “engineered” to bind to apredetermined nucleotide sequence. Non-limiting examples of methods forengineering zinc finger proteins are design and selection. A designedzinc finger protein is a protein not occurring in nature whosedesign/composition results principally from rational criteria. Rationalcriteria for design include application of substitution rules andcomputerized algorithms for processing information in a database storinginformation of existing ZFP designs and binding data. See, for example,U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

A “selected” zinc finger protein is a protein not found in nature whoseproduction results primarily from an empirical process such as phagedisplay, interaction trap or hybrid selection. See e.g., U.S. Pat. No.5,789,538; U.S. Pat. No. 5,925,523; U.S. Pat. No. 6,007,988; U.S. Pat.No. 6,013,453; U.S. Pat. No. 6,200,759; WO 95/19431; WO 96/06166; WO98/53057; WO 98/54311; WO 00/27878; WO 01/60970 WO 01/88197 and WO02/099084.

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides are used for targeted double-strandedDNA cleavage.

A “cleavage half-domain” is a polypeptide sequence which, in conjunctionwith a second polypeptide (either identical or different) forms acomplex having cleavage activity (preferably double-strand cleavageactivity). The terms “first and second cleavage half-domains;” “+ and −cleavage half-domains” and “right and left cleavage half-domains” areused interchangeably to refer to pairs of cleavage half-domains thatdimerize.

An “engineered cleavage half-domain” is a cleavage half-domain that hasbeen modified so as to form obligate heterodimers with another cleavagehalf-domain (e.g., another engineered cleavage half-domain). See, also,U.S. patent application Ser. Nos. 10/912,932 and 11/304,981 and U.S.Provisional Application No. 60/808,486 (filed May 25, 2006),incorporated herein by reference in their entireties.

The term “sequence” refers to a nucleotide sequence of any length, whichcan be DNA or RNA; can be linear, circular or branched and can be eithersingle-stranded or double stranded. The term “donor sequence” refers toa nucleotide sequence that is inserted into a genome. A donor sequencecan be of any length, for example between 2 and 10,000 nucleotides inlength (or any integer value there between or there above), preferablybetween about 100 and 1,000 nucleotides in length (or any integer therebetween), more preferably between about 200 and 500 nucleotides inlength. “Chromatin” is the nucleoprotein structure comprising thecellular genome. Cellular chromatin comprises nucleic acid, primarilyDNA, and protein, including histones and non-histone chromosomalproteins. The majority of eukaryotic cellular chromatin exists in theform of nucleosomes, wherein a nucleosome core comprises approximately150 base pairs of DNA associated with an octamer comprising two each ofhistones H2A, H2B, H3 and H4; and linker DNA (of variable lengthdepending on the organism) extends between nucleosome cores. A moleculeof histone H1 is generally associated with the linker DNA. For thepurposes of the present disclosure, the term “chromatin” is meant toencompass all types of cellular nucleoprotein, both prokaryotic andeukaryotic. Cellular chromatin includes both chromosomal and episomalchromatin.

A “chromosome,” is a chromatin complex comprising all or a portion ofthe genome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist. For example, thesequence 5′-GAATTC-3′ is a target site for the Eco RI restrictionendonuclease.

A “chronic infectious disease” is a disease caused by an infectiousagent wherein the infection has persisted. Such a disease may includehepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-1, HSV-6, HSV-II,CMV, and EBV), and HIV/AIDS. Non-viral examples may include chronicfungal diseases such Aspergillosis, Candidiasis, Coccidioidomycosis, anddiseases associated with Cryptococcus and Histoplasmosis. None limitingexamples of chronic bacterial infectious agents may be Chlamydiapneumoniae, Listeria monocytogenes, and Mycobacterium tuberculosis. Theterm “cancer” refers to any disease in which there is an unrestrainedproliferation of cells, either within an organ or body tissue. Thus, theterm includes any type of cancer or malignancy, including, but notlimited to, ovarian cancer, leukemia, lung cancer, colorectal/coloncancer, CNS cancer, melanoma, renal cell carcinoma,plasmacytoma/myeloma, prostate cancer, breast cancer, and the like. Asused herein, the term “tumor” refers to an abnormal growth of cells ortissues of the malignant type, unless otherwise specifically indicatedand does not include a benign type tissue. The term “inhibits orinhibiting” as used herein means reducing growth/replication.

The term “autoimmune disease” refers to any disease or disorder in whichthe subject mounts a destructive immune response against its owntissues. Autoimmune disorders can affect almost every organ system inthe subject (e.g., human), including, but not limited to, diseases ofthe nervous, gastrointestinal, and endocrine systems, as well as skinand other connective tissues, eyes, blood and blood vessels. Examples ofautoimmune diseases include, but are not limited to Hashimoto'sthyroiditis, Systemic lupus erythematosus, Sjogren's syndrome, Graves'disease, Scleroderma, Rheumatoid arthritis, Multiple sclerosis,Myasthenia gravis and Diabetes.

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPDNA-binding domain and a cleavage domain) and fusion nucleic acids (forexample, a nucleic acid encoding the fusion protein described supra).Examples of the second type of fusion molecule include, but are notlimited to, a fusion between a triplex-forming nucleic acid and apolypeptide, and a fusion between a minor groove binder and a nucleicacid.

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of an mRNA. Gene products also include RNAswhich are modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

“Modulation” of gene expression refers to a change in the expressionlevel of a gene. Modulation of expression can include, but is notlimited to, gene activation and gene repression. Modulation may also becomplete, i.e. wherein gene expression is totally inactivated or isactivated to wildtype levels or beyond; or it may be partial, whereingene expression is partially reduced, or partially activated to somefraction of wildtype levels. “Eukaryotic” cells include, but are notlimited to, fungal cells (such as yeast), plant cells, animal cells,mammalian cells and human cells (e.g., T-cells).

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a ZFPDNA-binding domain is fused to a cleavage domain, the ZFP DNA-bindingdomain and the cleavage domain are in operative linkage if, in thefusion polypeptide, the ZFP DNA-binding domain portion is able to bindits target site and/or its binding site, while the cleavage domain isable to cleave DNA in the vicinity of the target site. Similarly, withrespect to a fusion polypeptide in which a ZFP DNA-binding domain isfused to an activation or repression domain, the ZFP DNA-binding domainand the activation or repression domain are in operative linkage if, inthe fusion polypeptide, the ZFP DNA-binding domain portion is able tobind its target site and/or its binding site, while the activationdomain is able to upregulate gene expression or the repression domain isable to downregulate gene expression.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain oneore more amino acid or nucleotide substitutions. Methods for determiningthe function of a nucleic acid (e.g., coding function, ability tohybridize to another nucleic acid) are well-known in the art. Similarly,methods for determining protein function are well-known. For example,the DNA-binding function of a polypeptide can be determined, forexample, by filter-binding, electrophoretic mobility-shift, orimmunoprecipitation assays. DNA cleavage can be assayed by gelelectrophoresis. See Ausubel et al., supra. The ability of a protein tointeract with another protein can be determined, for example, byco-immunoprecipitation, two-hybrid assays or complementation, bothgenetic and biochemical. See, for example, Fields et al. (1989) Nature340:245-246; U.S. Pat. No. 5,585,245 and PCT WO 98/44350.

A “vector” is capable of transferring gene sequences to target cells.Typically, “vector construct,” “expression vector,” and “gene transfervector,” mean any nucleic acid construct capable of directing theexpression of a gene of interest and which can transfer gene sequencesto target cells. Thus, the term includes cloning, and expressionvehicles, as well as integrating vectors.

A “reporter gene” or “reporter sequence” refers to any sequence thatproduces a protein product that is easily measured, preferably althoughnot necessarily in a routine assay. Suitable reporter genes include, butare not limited to, sequences encoding proteins that mediate antibioticresistance (e.g., ampicillin resistance, neomycin resistance, G418resistance, puromycin resistance), sequences encoding colored orfluorescent or luminescent proteins (e.g., green fluorescent protein,enhanced green fluorescent protein, red fluorescent protein,luciferase), and proteins which mediate enhanced cell growth and/or geneamplification (e.g., dihydrofolate reductase). Epitope tags include, forexample, one or more copies of FLAG, His, myc, Tap, HA or any detectableamino acid sequence. “Expression tags” include sequences that encodereporters that may be operably linked to a desired gene sequence inorder to monitor expression of the gene of interest.

Overview

Described herein are zinc finger protein-transcription factors (ZFP-TFs)and/or nucleases (e.g., ZFNs) targeted to the PD1 gene as well ascompositions comprising and methods of using these ZFP-TFs and/ornucleases for treatment of disease or disorders in which PD1 isaberrantly or undesirably expressed, or wherein the PD1 pathway isaberrantly or undesirably utilized due to overexpression of a PD1ligand, including, for example, treatment of chronic infectiousdiseases, cancers, and/or autoimmmune diseases. For treatment of asubject with a disease or disorder that is ameliorated by the modulationof PD1 expression, the ZFP-TFs and/or nucleases described herein can beintroduced in vivo or ex vivo into cells (e.g., primary cells isolatedfrom a patient afflicted with such a disease). Following ZFP-TF and/orZFN treatment, the cells may be reintroduced into the patient for use asa medicament in the treatment of a chronic infectious disease or cancer.Similarly, stem cells may be used that have been treated with thePD1-specific ZFNs and/or ZFP-TFs. These cells can be infused into anafflicted patient for treatment of such a medical condition.

The compositions and methods described herein thus allow for themodulation of a PD1 gene. PD1 expression may be knocked out, knockeddown, upregulated or down-regulated using PD1-specific ZFP TFs or ZFNs,depending on the need. PD1 expression may be downregulated with ZFP-TFsor one or more PD1-specific ZFNs, for example in patients afflicted withchronic infectious diseases or cancers, and may be upregulated inpatients, for example patients with autoimmune disease. The methods andcompositions of the invention also provide therapeutics comprising apolynucleotide encoding the PD1-specific ZFP TFs and/or nucleases,wherein the polynucleotide is administered directly to the patient.Further, the invention provides methods and compositions wherein thepolynucleotide encoding the ZFP TFs and/or nucleases may be incorporatedinto a vector such as a viral delivery vehicle for systemicadministration as a therapeutic to an afflicted patient.

DNA-Binding Domains

Described herein are compositions comprising a DNA-binding domain thatspecifically binds to a target site in a PD1 gene. Any DNA-bindingdomain can be used in the compositions and methods disclosed herein,including but not limited to a zinc finger DNA-binding domain or aDNA-binding domain from a meganuclease.

In certain embodiments, the DNA binding domain comprises a zinc fingerprotein. Preferably, the zinc finger protein is non-naturally occurringin that it is engineered to bind to a target site of choice. See, forexample, Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo et al.(2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) NatureBiotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol.12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416;U.S. Pat. Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558;7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635;7,253,273; and U.S. Patent Publication Nos. 2005/0064474; 2007/0218528;2005/0267061, all incorporated herein by reference in their entireties.

An engineered zinc finger binding domain can have a novel bindingspecificity, compared to a naturally-occurring zinc finger protein.Engineering methods include, but are not limited to, rational design andvarious types of selection. Rational design includes, for example, usingdatabases comprising triplet (or quadruplet) nucleotide sequences andindividual zinc finger amino acid sequences, in which each triplet orquadruplet nucleotide sequence is associated with one or more amino acidsequences of zinc fingers which bind the particular triplet orquadruplet sequence. See, for example, co-owned U.S. Pat. Nos. 6,453,242and 6,534,261, incorporated by reference herein in their entireties.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB2,338,237. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in co-owned WO02/077227.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in co-owned WO02/077227.

Selection of target sites; ZFPs and methods for design and constructionof fusion proteins (and polynucleotides encoding same) are known tothose of skill in the art and described in detail in U.S. Pat. Nos.6,140,0815; 789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988;6,013,453; 6,200,759; WO 95/19431; WO 96/06166; WO 98/53057; WO98/54311; WO 00/27878; WO 01/60970 WO 01/88197; WO 02/099084; WO98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein.

Alternatively, the DNA-binding domain may be derived from a nuclease.For example, the recognition sequences of homing endonucleases andmeganucleases such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI,I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIIIare known. See also U.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252;Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al.(1989) Gene 82:115-118; Perler et al. (1994) Nucleic Acids Res. 22,1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al. (1996)J. Mol. Biol. 263:163-180; Argast et al. (1998) J. Mol. Biol.280:345-353 and the New England Biolabs catalogue. In addition, theDNA-binding specificity of homing endonucleases and meganucleases can beengineered to bind non-natural target sites. See, for example, Chevalieret al. (2002) Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic AcidsRes. 31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques etal. (2007) Current Gene Therapy 7:49-66; U.S. Patent Publication No.20070117128.

In certain embodiments, the DNA binding domain is an engineered zincfinger protein that binds (in a sequence-specific manner) to a targetsite in a PD1 gene and modulates expression of PD1. The ZFPs can bindselectively to either a mutant PD1 allele or a wildtype PD1 sequence.PD1 target sites typically include at least one zinc finger but caninclude a plurality of zinc fingers (e.g., 2, 3, 4, 5, 6 or morefingers). Usually, the ZFPs include at least three fingers. Certain ofthe ZFPs include four, five or six fingers. The ZFPs that include threefingers typically recognize a target site that includes 9 or 10nucleotides; ZFPs that include four fingers typically recognize a targetsite that includes 12 to 14 nucleotides; while ZFPs having six fingerscan recognize target sites that include 18 to 21 nucleotides. The ZFPscan also be fusion proteins that include one or more regulatory domains,wherein these regulatory domains can be transcriptional activation orrepression domains.

In some embodiments, the DNA binding domain is an engineered domain froma TAL effector derived from the plant pathogen Xanthomonas (see Boch etal, (2009) Science 29 Oct. 2009 (10.1126/science.117881) and Moscou andBogdanove, (2009) Science 29 Oct. 2009 (10.1126/science.1178817).

Fusion Proteins

Fusion proteins comprising DNA-binding proteins (e.g., ZFPs) asdescribed herein and a heterologous regulatory (functional) domain (orfunctional fragment thereof) are also provided. Common domains include,e.g., transcription factor domains (activators, repressors,co-activators, co-repressors), silencers, oncogenes (e.g., myc, jun,fos, myb, max, mad, rel, ets, bcl, myb, mos family members etc.); DNArepair enzymes and their associated factors and modifiers; DNArearrangement enzymes and their associated factors and modifiers;chromatin associated proteins and their modifiers (e g kinases,acetylases and deacetylases); and DNA modifying enzymes (e.g.,methyltransferases, topoisomerases, helicases, ligases, kinases,phosphatases, polymerases, endonucleases) and their associated factorsand modifiers. U.S. Patent Application Publication Nos. 20050064474;20060188987 and 2007/0218528 for details regarding fusions ofDNA-binding domains and nuclease cleavage domains, incorporated byreference in their entireties herein.

Suitable domains for achieving activation include the HSV VP16activation domain (see, e.g., Hagmann et al., J. Virol. 71, 5952-5962(1997)) nuclear hormone receptors (see, e.g., Torchia et al., Curr.Opin. Cell. Biol. 10:373-383 (1998)); the p65 subunit of nuclear factorkappa B (Bitko & Batik, J. Virol. 72:5610-5618 (1998) and Doyle & Hunt,Neuroreport 8:2937-2942 (1997)); Liu et al., Cancer Gene Ther. 5:3-28(1998)), or artificial chimeric functional domains such as VP64 (Beerliet al., (1998) Proc. Natl. Acad. Sci. USA 95:14623-33), and degron(Molinari et al., (1999) EMBO J. 18, 6439-6447). Additional exemplaryactivation domains include, Oct 1, Oct-2A, Sp1, AP-2, and CTF1 (Seipelet al., EMBO J. 11, 4961-4968 (1992) as well as p300, CBP, PCAF, SRC1PvALF, AtHD2A and ERF-2. See, for example, Robyr et al. (2000) Mol.Endocrinol. 14:329-347; Collingwood et al. (1999) J. Mol. Endocrinol.23:255-275; Leo et al. (2000) Gene 245:1-11; Manteuffel-Cymborowska(1999) Acta Biochim. Pol. 46:77-89; McKenna et al. (1999) J. SteroidBiochem. Mol. Biol. 69:3-12; Malik et al. (2000) Trends Biochem. Sci.25:277-283; and Lemon et al. (1999) Curr. Opin. Genet. Dev. 9:499-504.Additional exemplary activation domains include, but are not limited to,OsGAI, HALF-1, C1, AP1, ARF-5, -6, -7, and -8, CPRF1, CPRF4, MYC-RP/GP,and TRAB1. See, for example, Ogawa et al. (2000) Gene 245:21-29; Okanamiet al. (1996) Genes Cells 1:87-99; Goff et al. (1991) Genes Dev.5:298-309; Cho et al. (1999) Plant Mol. Biol. 40:419-429; Ulmason et al.(1999) Proc. Natl. Acad. Sci. USA 96:5844-5849; Sprenger-Haussels et al.(2000) Plant J. 22:1-8; Gong et al. (1999) Plant Mol. Biol. 41:33-44;and Hobo et al. (1999) Proc. Natl. Acad. Sci. USA 96:15,348-15,353.

It will be clear to those of skill in the art that, in the formation ofa fusion protein (or a nucleic acid encoding same) between a DNA-bindingdomain and a functional domain, either an activation domain or amolecule that interacts with an activation domain is suitable as afunctional domain. Essentially any molecule capable of recruiting anactivating complex and/or activating activity (such as, for example,histone acetylation) to the target gene is useful as an activatingdomain of a fusion protein. Insulator domains, localization domains, andchromatin remodeling proteins such as ISWI-containing domains and/ormethyl binding domain proteins suitable for use as functional domains infusion molecules are described, for example, in co-owned U.S. PatentApplications 2002/0115215 and 2003/0082552 and in co-owned WO 02/44376.

Exemplary repression domains include, but are not limited to, KRAB A/B,KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3,members of the DNMT family (e.g., DNMT1, DNMT3A, DNMT3B), Rb, and MeCP2.See, for example, Bird et al. (1999) Cell 99:451-454; Tyler et al.(1999) Cell 99:443-446; Knoepfler et al. (1999) Cell 99:447-450; andRobertson et al. (2000) Nature Genet. 25:338-342. Additional exemplaryrepression domains include, but are not limited to, ROM2 and AtHD2A.See, for example, Chem et al. (1996) Plant Cell 8:305-321; and Wu et al.(2000) Plant J. 22:19-27.

Fusion molecules are constructed by methods of cloning and biochemicalconjugation that are well known to those of skill in the art. Fusionmolecules comprise a DNA-binding domain and a functional domain (e.g., atranscriptional activation or repression domain). Fusion molecules alsooptionally comprise nuclear localization signals (such as, for example,that from the SV40 medium T-antigen) and epitope tags (such as, forexample, FLAG and hemagglutinin) Fusion proteins (and nucleic acidsencoding them) are designed such that the translational reading frame ispreserved among the components of the fusion.

Fusions between a polypeptide component of a functional domain (or afunctional fragment thereof) on the one hand, and a non-proteinDNA-binding domain (e.g., antibiotic, intercalator, minor groove binder,nucleic acid) on the other, are constructed by methods of biochemicalconjugation known to those of skill in the art. See, for example, thePierce Chemical Company (Rockford, Ill.) Catalogue. Methods andcompositions for making fusions between a minor groove binder and apolypeptide have been described. Mapp et al. (2000) Proc. Natl. Acad.Sci. USA 97:3930-3935.

In certain embodiments, the target site bound by the zinc finger proteinis present in an accessible region of cellular chromatin. Accessibleregions can be determined as described, for example, in co-ownedInternational Publication WO 01/83732. If the target site is not presentin an accessible region of cellular chromatin, one or more accessibleregions can be generated as described in co-owned WO 01/83793. Inadditional embodiments, the DNA-binding domain of a fusion molecule iscapable of binding to cellular chromatin regardless of whether itstarget site is in an accessible region or not. For example, suchDNA-binding domains are capable of binding to linker DNA and/ornucleosomal DNA. Examples of this type of “pioneer” DNA binding domainare found in certain steroid receptor and in hepatocyte nuclear factor 3(HNF3). Cordingley et al. (1987) Cell 48:261-270; Pina et al. (1990)Cell 60:719-731; and Cirillo et al. (1998) EMBO J. 17:244-254.

The fusion molecule may be formulated with a pharmaceutically acceptablecarrier, as is known to those of skill in the art. See, for example,Remington's Pharmaceutical Sciences, 17th ed., 1985; and co-owned WO00/42219.

The functional component/domain of a fusion molecule can be selectedfrom any of a variety of different components capable of influencingtranscription of a gene once the fusion molecule binds to a targetsequence via its DNA binding domain. Hence, the functional component caninclude, but is not limited to, various transcription factor domains,such as activators, repressors, co-activators, co-repressors, andsilencers.

Additional exemplary functional domains are disclosed, for example, inco-owned U.S. Pat. No. 6,534,261 and US Patent Application PublicationNo. 2002/0160940.

Functional domains that are regulated by exogenous small molecules orligands may also be selected. For example, RheoSwitch® technology may beemployed wherein a functional domain only assumes its activeconformation in the presence of the external RheoChem™ ligand (see forexample US 20090136465). Thus, the ZFP may be operably linked to theregulatable functional domain wherein the resultant activity of theZFP-TF is controlled by the external ligand. In certain embodiments, thefusion protein comprises a DNA-binding binding domain and cleavage(nuclease) domain. As such, gene modification can be achieved using anuclease, for example an engineered nuclease. Engineered nucleasetechnology is based on the engineering of naturally occurringDNA-binding proteins. The methods and compositions described herein arebroadly applicable and may involve any nuclease of interest.Non-limiting examples of nucleases include meganucleases and zinc fingernucleases. The nuclease may comprise heterologous DNA-binding andcleavage domains (e.g., zinc finger nucleases; meganuclease DNA-bindingdomains with heterologous cleavage domains) or, alternatively, theDNA-binding domain of a naturally-occurring nuclease may be altered tobind to a selected target site (e.g., a meganuclease that has beenengineered to bind to site different than the cognate binding site). Forexample, engineering of homing endonucleases with tailored DNA-bindingspecificities has been described, see Chames et al. (2005) Nucleic AcidsRes 33(20):e178; Arnould et al. (2006) J. Mol. Biol. 355:443-458 andGrizot et at (2009) Nucleic Acids Res July 7 e publication. In addition,engineering of ZFPs has also been described. See, e.g., U.S. Pat. Nos.6,534,261; 6,607,882; 6,824,978; 6,979,539; 6,933,113; 7,163,824; and7,013,219.

In certain embodiment, the nuclease is a meganuclease (homingendonuclease). Naturally-occurring meganucleases recognize 15-40base-pair cleavage sites and are commonly grouped into four families:the LAGLIDADG (SEQ ID NO: 79) family, the GIY-YIG family, the His-Cystbox family and the HNH family. Exemplary homing endonucleases includeI-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII,I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII. Theirrecognition sequences are known. See also U.S. Pat. No. 5,420,032; U.S.Pat. No. 6,833,252; Belfort et al. (1997) Nucleic Acids Res.25:3379-3388; Dujon et al. (1989) Gene 82:115-118; Perler et al. (1994)Nucleic Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet. 12:224-228;Gimble et al. (1996) J. Mol. Biol. 263:163-180; Argast et al. (1998) J.Mol. Biol. 280:345-353 and the New England Biolabs catalogue.

DNA-binding domains from naturally-occurring meganucleases, primarilyfrom the LAGLIDADG (SEQ ID NO: 79) family, have been used to promotesite-specific genome modification in plants, yeast, Drosophila,mammalian cells and mice, but this approach has been limited to themodification of either homologous genes that conserve the meganucleaserecognition sequence (Monet et al. (1999), Biochem. Biophysics. Res.Common. 255: 88-93) or to pre-engineered genomes into which arecognition sequence has been introduced (Route et al. (1994), Mol.Cell. Biol. 14: 8096-106; Chilton et al. (2003), Plant Physiology. 133:956-65; Puchta et al. (1996), Proc. Natl. Acad. Sci. USA 93: 5055-60;Rong et al. (2002), Genes Dev. 16: 1568-81; Gouble et al. (2006), J.Gene Med. 8(5):616-622). Accordingly, attempts have been made toengineer meganucleases to exhibit novel binding specificity at medicallyor biotechnologically relevant sites (Porteus et al. (2005), Nat.Biotechnol. 23: 967-73; Sussman et al. (2004), J. Mol. Biol. 342: 31-41;Epinat et al. (2003), Nucleic Acids Res. 31: 2952-62; Chevalier et al.(2002) Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic Acids Res.31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques et al.(2007) Current Gene Therapy 7:49-66; U.S. Patent Publication Nos.20070117128; 20060206949; 20060153826; 20060078552; and 20040002092). Inaddition, naturally-occurring or engineered DNA-binding domains frommeganucleases have also been operably linked with a cleavage domain froma heterologous nuclease (e.g., FokI).

In other embodiments, the nuclease is a zinc finger nuclease (ZFN). ZFNscomprise a zinc finger protein that has been engineered to bind to atarget site in a gene of choice and cleavage domain or a cleavagehalf-domain.

As noted above, zinc finger binding domains can be engineered to bind toa sequence of choice. See, for example, Beerli et al. (2002) NatureBiotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem.70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal etal. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr.Opin. Struct. Biol. 10:411-416. An engineered zinc finger binding domaincan have a novel binding specificity, compared to a naturally-occurringzinc finger protein. Engineering methods include, but are not limitedto, rational design and various types of selection. Rational designincludes, for example, using databases comprising triplet (orquadruplet) nucleotide sequences and individual zinc finger amino acidsequences, in which each triplet or quadruplet nucleotide sequence isassociated with one or more amino acid sequences of zinc fingers whichbind the particular triplet or quadruplet sequence. See, for example,co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261, incorporated byreference herein in their entireties.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB2,338,237. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in co-owned WO02/077227.

Selection of target sites; ZFNs and methods for design and constructionof fusion proteins (and polynucleotides encoding same) are known tothose of skill in the art and described in detail in U.S. PatentApplication Publication Nos. 20050064474 and 20060188987, incorporatedby reference in their entireties herein.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, e.g., U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein.

Nucleases such as ZFNs and/or meganucleases also comprise a nuclease(cleavage domain, cleavage half-domain). As noted above, the cleavagedomain may be heterologous to the DNA-binding domain, for example a zincfinger DNA-binding domain and a cleavage domain from a nuclease or ameganuclease DNA-binding domain and cleavage domain from a differentnuclease. Heterologous cleavage domains can be obtained from anyendonuclease or exonuclease. Exemplary endonucleases from which acleavage domain can be derived include, but are not limited to,restriction endonucleases and homing endonucleases. See, for example,2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort etal. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes whichcleave DNA are known (e.g., 51 Nuclease; mung bean nuclease; pancreaticDNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn etal. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One ormore of these enzymes (or functional fragments thereof) can be used as asource of cleavage domains and cleavage half-domains.

Similarly, a cleavage half-domain can be derived from any nuclease orportion thereof, as set forth above, that requires dimerization forcleavage activity. In general, two fusion proteins are required forcleavage if the fusion proteins comprise cleavage half-domains.Alternatively, a single protein comprising two cleavage half-domains canbe used. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof). In addition, the target sites for the two fusionproteins are preferably disposed, with respect to each other, such thatbinding of the two fusion proteins to their respective target sitesplaces the cleavage half-domains in a spatial orientation to each otherthat allows the cleavage half-domains to form a functional cleavagedomain, e.g., by dimerizing. Thus, in certain embodiments, the nearedges of the target sites are separated by 5-8 nucleotides or by 15-18nucleotides. However any integral number of nucleotides or nucleotidepairs can intervene between two target sites (e.g., from 2 to 50nucleotide pairs or more). In general, the site of cleavage lies betweenthe target sites.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme Fok I catalyzesdouble-stranded cleavage of DNA, at 9 nucleotides from its recognitionsite on one strand and 13 nucleotides from its recognition site on theother. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768;Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al.(1994b) J. Biol. Chem. 269:31,978-31,982. Thus, in one embodiment,fusion proteins comprise the cleavage domain (or cleavage half-domain)from at least one Type IIS restriction enzyme and one or more zincfinger binding domains, which may or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is Fok I. This particular enzyme isactive as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA95: 10,570-10,575. Accordingly, for the purposes of the presentdisclosure, the portion of the Fok I enzyme used in the disclosed fusionproteins is considered a cleavage half-domain. Thus, for targeteddouble-stranded cleavage and/or targeted replacement of cellularsequences using zinc finger-Fok I fusions, two fusion proteins, eachcomprising a FokI cleavage half-domain, can be used to reconstitute acatalytically active cleavage domain. Alternatively, a singlepolypeptide molecule containing a zinc finger binding domain and two FokI cleavage half-domains can also be used. Parameters for targetedcleavage and targeted sequence alteration using zinc finger-Fok Ifusions are provided elsewhere in this disclosure.

A cleavage domain or cleavage half-domain can be any portion of aprotein that retains cleavage activity, or that retains the ability tomultimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary Type IIS restriction enzymes are described in InternationalPublication WO 07/014,275, incorporated herein in its entirety.Additional restriction enzymes also contain separable binding andcleavage domains, and these are contemplated by the present disclosure.See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

In certain embodiments, the cleavage domain comprises one or moreengineered cleavage half-domain (also referred to as dimerization domainmutants) that minimize or prevent homodimerization, as described, forexample, in U.S. Patent Publication Nos. 20050064474 and 20060188987 andin U.S. application Ser. No. 11/805,850 (filed May 23, 2007), thedisclosures of all of which are incorporated by reference in theirentireties herein. Amino acid residues at positions 446, 447, 479, 483,484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 ofFok I are all targets for influencing dimerization of the Fok I cleavagehalf-domains.

Exemplary engineered cleavage half-domains of Fok I that form obligateheterodimers include a pair in which a first cleavage half-domainincludes mutations at amino acid residues at positions 490 and 538 ofFok I and a second cleavage half-domain includes mutations at amino acidresidues 486 and 499.

Thus, in one embodiment, a mutation at 490 replaces Glu (E) with Lys(K); the mutation at 538 replaces Iso (I) with Lys (K); the mutation at486 replaced Gln (Q) with Glu (E); and the mutation at position 499replaces Iso (I) with Lys (K). Specifically, the engineered cleavagehalf-domains described herein were prepared by mutating positions 490(E→K) and 538 (I→K) in one cleavage half-domain to produce an engineeredcleavage half-domain designated “E490K:I538K” and by mutating positions486 (Q→E) and 499 (I→L) in another cleavage half-domain to produce anengineered cleavage half-domain designated “Q486E:I499L”. The engineeredcleavage half-domains described herein are obligate heterodimer mutantsin which aberrant cleavage is minimized or abolished. See, e.g., Example1 of U.S. Provisional Application No. 60/808,486 (filed May 25, 2006),the disclosure of which is incorporated by reference in its entirety forall purposes.

Engineered cleavage half-domains described herein can be prepared usingany suitable method, for example, by site-directed mutagenesis ofwild-type cleavage half-domains (Fok I) as described in Example 5 ofU.S. Patent Publication No. 20050064474 and Example 38 of U.S. PatentPublication Nos. 2007/0305346 and 2008/0131962 and U.S. PatentProvisional Application Nos. 61/337,769, filed Feb. 8, 2010 and61/403,916, filed Sep. 23, 2010.

Nuclease expression constructs can be readily designed using methodsknown in the art. See, e.g., United States Patent Publications20030232410; 20050208489; 20050026157; 20050064474; 20060188987;20060063231; and International Publication WO 07/014,275. In certainembodiments, expression of the nuclease is under the control of aninducible promoter, for example the galactokinase promoter which isactivated (de-repressed) in the presence of raffinose and/or galactoseand repressed in presence of glucose. In particular, the galactokinasepromoter is induced and the nuclease(s) expressed upon successivechanges in the carbon source (e.g., from glucose to raffinose togalactose). Other non-limiting examples of inducible promoters includeCUP1, MET15, PHO5, and tet-responsive promoters.

Delivery

The proteins (e.g., ZFPs), polynucleotides encoding same andcompositions comprising the proteins and/or polynucleotides describedherein may be delivered to a target cell by any suitable means. Suitablecells include but not limited to eukaryotic and prokaryotic cells and/orcell lines. Non-limiting examples of such cells or cell lines generatedfrom such cells include COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44,CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK,HaK, NS0, SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T),and perC6 cells as well as insect cells such as Spodoptera fugiperda(Sf), or fungal cells such as Saccharomyces, Pichia andSchizosaccharomyces. In certain embodiments, the cell line is a CHO-K1,MDCK or HEK293 cell line. Suitable primary cells include peripheralblood mononuclear cells (PBMC), and other blood cell subsets such as,but not limited to, CD4+ T cells or CD8+ T cells. Suitable cells alsoinclude stem cells such as, by way of example, embryonic stem cells,induced pluripotent stem cells, hematopoietic stem cells, neuronal stemcells and mesenchymal stem cells.

Methods of delivering proteins comprising zinc finger proteins asdescribed herein are described, for example, in U.S. Pat. Nos.6,453,242; 6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558;6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, thedisclosures of all of which are incorporated by reference herein intheir entireties.

Zinc finger proteins as described herein may also be delivered usingvectors containing sequences encoding one or more of the zinc fingerprotein(s). Any vector systems may be used including, but not limitedto, plasmid vectors, retroviral vectors, lentiviral vectors, adenovirusvectors, poxvirus vectors; herpesvirus vectors and adeno-associatedvirus vectors, etc. See, also, U.S. Pat. Nos. 6,534,261; 6,607,882;6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, incorporatedby reference herein in their entireties. Furthermore, it will beapparent that any of these vectors may comprise one or more zinc fingerprotein-encoding sequences. Thus, when one or more ZFPs are introducedinto the cell, the ZFPs may be carried on the same vector or ondifferent vectors. When multiple vectors are used, each vector maycomprise a sequence encoding one or multiple ZFPs.

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding engineered ZFPs in cells (e.g.,mammalian cells) and target tissues. Such methods can also be used toadminister nucleic acids encoding ZFPs to cells in vitro. In certainembodiments, nucleic acids encoding ZFPs are administered for in vivo orex vivo gene therapy uses. Non-viral vector delivery systems include DNAplasmids, naked nucleic acid, and nucleic acid complexed with a deliveryvehicle such as a liposome or poloxamer. Viral vector delivery systemsinclude DNA and RNA viruses, which have either episomal or integratedgenomes after delivery to the cell. For a review of gene therapyprocedures, see Anderson, Science 256:808-813 (1992); Nabel & Feigner,TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993);Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992);Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, RestorativeNeurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, BritishMedical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topicsin Microbiology and Immunology Doerfler and Bohm (eds.) (1995); and Yuet al., Gene Therapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids include electroporation,lipofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Sonoporationusing, e.g., the Sonitron 2000 system (Rich-Mar) can also be used fordelivery of nucleic acids.

Additional exemplary nucleic acid delivery systems include thoseprovided by Amaxa® Biosystems (Cologne, Germany), Maxcyte, Inc.(Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) andCopernicus Therapeutics Inc, (see for example U.S. Pat. No. 6,008,336).Lipofection is described in e.g., U.S. Pat. No. 5,049,386, U.S. Pat. No.4,946,787; and U.S. Pat. No. 4,897,355) and lipofection reagents aresold commercially (e.g., Transfectam™ and Lipofectin™). Cationic andneutral lipids that are suitable for efficient receptor-recognitionlipofection of polynucleotides include those of Feigner, WO 91/17424, WO91/16024. Delivery can be to cells (ex vivo administration) or targettissues (in vivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

Additional methods of delivery include the use of packaging the nucleicacids to be delivered into EnGeneIC delivery vehicles (EDVs). These EDVsare specifically delivered to target tissues using bispecific antibodieswhere one arm of the antibody has specificity for the target tissue andthe other has specificity for the EDV. The antibody brings the EDVs tothe target cell surface and then the EDV is brought into the cell byendocytosis. Once in the cell, the contents are released (see MacDiamidet at (2009) Nature Biotechnology vol 27(7) p. 643).

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding engineered ZFPs take advantage of highly evolvedprocesses for targeting a virus to specific cells in the body andtrafficking the viral payload to the nucleus. Viral vectors can beadministered directly to patients (in vivo) or they can be used to treatcells in vitro and the modified cells are administered to patients (exvivo). Conventional viral based systems for the delivery of ZFPsinclude, but are not limited to, retroviral, lentivirus, adenoviral,adeno-associated, vaccinia and herpes simplex virus vectors for genetransfer. Integration in the host genome is possible with theretrovirus, lentivirus, and adeno-associated virus gene transfermethods, often resulting in long term expression of the insertedtransgene. Additionally, high transduction efficiencies have beenobserved in many different cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system depends on thetarget tissue. Retroviral vectors are comprised of cis-acting longterminal repeats with packaging capacity for up to 6-10 kb of foreignsequence. The minimum cis-acting LTRs are sufficient for replication andpackaging of the vectors, which are then used to integrate thetherapeutic gene into the target cell to provide permanent transgeneexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmunodeficiency virus (SIV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications in which transient expression is preferred, adenoviralbased systems can be used. Adenoviral based vectors are capable of veryhigh transduction efficiency in many cell types and do not require celldivision. With such vectors, high titer and high levels of expressionhave been obtained. This vector can be produced in large quantities in arelatively simple system. Adeno-associated virus (“AAV”) vectors arealso used to transduce cells with target nucleic acids, e.g., in the invitro production of nucleic acids and peptides, and for in vivo and exvivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47(1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994).Construction of recombinant AAV vectors are described in a number ofpublications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol.Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol.4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989).

At least six viral vector approaches are currently available for genetransfer in clinical trials, which utilize approaches that involvecomplementation of defective vectors by genes inserted into helper celllines to generate the transducing agent.

pLASN and MFG-S are examples of retroviral vectors that have been usedin clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn etal., Nat. Med. 1:1017-102 (1995); Malech et al., PNAS 94:22 12133-12138(1997)). PA317/pLASN was the first therapeutic vector used in a genetherapy trial. (Blaese et al., Science 270:475-480 (1995)). Transductionefficiencies of 50% or greater have been observed for MFG-S packagedvectors. (Ellem et al., Immunol Immunother. 44(1):10-20 (1997); Dranoffet al., Hum. Gene Ther. 1:111-2 (1997).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther.9:748-55 (1996)). Other AAV serotypes, including AAV1, AAV3, AAV4, AAV5,AAV6 and AAV8, can also be used in accordance with the presentinvention.

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer and readily infect a number of different celltypes. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiple types oftissues in vivo, including nondividing, differentiated cells such asthose found in liver, kidney and muscle. Conventional Ad vectors have alarge carrying capacity. An example of the use of an Ad vector in aclinical trial involved polynucleotide therapy for antitumorimmunization with intramuscular injection (Sterman et al., Hum. GeneTher. 7:1083-9 (1998)). Additional examples of the use of adenovirusvectors for gene transfer in clinical trials include Rosenecker et al.,Infection 24:1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:71083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarezet al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther.5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by a producer cellline that packages a nucleic acid vector into a viral particle. Thevectors typically contain the minimal viral sequences required forpackaging and subsequent integration into a host (if applicable), otherviral sequences being replaced by an expression cassette encoding theprotein to be expressed. The missing viral functions are supplied intrans by the packaging cell line. For example, AAV vectors used in genetherapy typically only possess inverted terminal repeat (ITR) sequencesfrom the AAV genome which are required for packaging and integrationinto the host genome. Viral DNA is packaged in a cell line, whichcontains a helper plasmid encoding the other AAV genes, namely rep andcap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. Accordingly, a viral vector can be modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the outer surface of the virus. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han et al., Proc. Natl. Acad.Sci. USA 92:9747-9751 (1995), reported that Moloney murine leukemiavirus can be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other virus-target cell pairs, in which the target cell expresses areceptor and the virus expresses a fusion protein comprising a ligandfor the cell-surface receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., FAB or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences which favor uptake byspecific target cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Ex vivo cell transfection for diagnostics, research, or for gene therapy(e.g., via re-infusion of the transfected cells into the host organism)is well known to those of skill in the art. In a preferred embodiment,cells are isolated from the subject organism, transfected with a ZFPnucleic acid (gene or cDNA), and re-infused back into the subjectorganism (e.g., patient). Various cell types suitable for ex vivotransfection are well known to those of skill in the art (see, e.g.,Freshney et al., Culture of Animal Cells, A Manual of Basic Technique(3rd ed. 1994)) and the references cited therein for a discussion of howto isolate and culture cells from patients).

Suitable cells include but not limited to eukaryotic and prokaryoticcells and/or cell lines. Non-limiting examples of such cells or celllines generated from such cells include COS, CHO (e.g., CHO-S, CHO-K1,CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79,B14AF28-G3, BHK, HaK, NS0, SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F,HEK293-H, HEK293-T), and perC6 cells as well as insect cells such asSpodoptera fugiperda (Sf), or fungal cells such as Saccharomyces, Pichiaand Schizosaccharomyces. In certain embodiments, the cell line is aCHO-K1, MDCK or HEK293 cell line. Additionally, primary cells may beisolated and used ex vivo for reintroduction into the subject to betreated following treatment with the ZFNs. Suitable primary cellsinclude peripheral blood mononuclear cells (PBMC), and other blood cellsubsets such as, but not limited to, CD4+ T cells or CD8+ T cells.Suitable cells also include stem cells such as, by way of example,embryonic stem cells, induced pluripotent stem cells, hematopoietic stemcells, neuronal stem cells and mesenchymal stem cells.

In one embodiment, stem cells are used in ex vivo procedures for celltransfection and gene therapy. The advantage to using stem cells is thatthey can be differentiated into other cell types in vitro, or can beintroduced into a mammal (such as the donor of the cells) where theywill engraft in the bone marrow. Methods for differentiating CD34+ cellsin vitro into clinically important immune cell types using cytokinessuch a GM-CSF, IFN-γ and TNF-α are known (see Inaba et al., J. Exp. Med.176:1693-1702 (1992)).

Stem cells are isolated for transduction and differentiation using knownmethods. For example, stem cells are isolated from bone marrow cells bypanning the bone marrow cells with antibodies which bind unwanted cells,such as CD4+ and CD8+ (T cells), CD45+ (panB cells), GR-1(granulocytes), and Iad (differentiated antigen presenting cells) (seeInaba et al., J. Exp. Med. 176:1693-1702 (1992)).

Stem cells that have been modified may also be used in some embodiments.For example, stem cells that have been made resistant to apoptosis maybe used as therapeutic compositions where the stem cells also containthe ZFP TFs of the invention. Resistance to apoptosis may come about,for example, by knocking out BAX and/or BAK using BAX- or BAK-specificZFNs (see, U.S. patent application Ser. No. 12/456,043) in the stemcells, or those that are disrupted in a caspase, again using caspase-6specific ZFNs for example. These cells can be transfected with the ZFPTFs that are known to regulate mutant or wildtype PD1.

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic ZFP nucleic acids can also be administered directly to anorganism for transduction of cells in vivo. Alternatively, naked DNA canbe administered. Administration is by any of the routes normally usedfor introducing a molecule into ultimate contact with blood or tissuecells including, but not limited to, injection, infusion, topicalapplication and electroporation. Suitable methods of administering suchnucleic acids are available and well known to those of skill in the art,and, although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective reaction than another route.

Methods for introduction of DNA into hematopoietic stem cells aredisclosed, for example, in U.S. Pat. No. 5,928,638. Vectors useful forintroduction of transgenes into hematopoietic stem cells, e.g., CD34⁺cells, include adenovirus Type 35.

Vectors suitable for introduction of transgenes into immune cells (e.g.,T-cells) include non-integrating lentivirus vectors. See, for example,Ory et al. (1996) Proc. Natl. Acad. Sci. USA 93:11382-11388; Dull et al.(1998) J. Virol. 72:8463-8471; Zuffery et al. (1998) J. Virol.72:9873-9880; Follenzi et al. (2000) Nature Genetics 25:217-222.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositionsavailable, as described below (see, e.g., Remington's PharmaceuticalSciences, 17th ed., 1989).

Applications

The disclosed compositions and methods can be used for any applicationin which it is desired to modulate the expression of one or more PD1genes. In particular, these methods and compositions can be used wheremodulation of a PD 1 allele is desired, including but not limited to,therapeutic and research applications. The methods and compositions maybe used to treat chronic infectious diseases such as HIV/AIDS and HCV.In addition, the methods and compositions may be used to treat cancerssuch as melanoma, ovarian cancer, colorectal/colon cancer, renal cellcarcinoma, plasmacytoma/myeloma, breast cancer and lung cancer.

Diseases and conditions which PD1 repressing ZFP TFs or ZFNs can be usedas therapeutic agents include, but are not limited to, chronicinfectious diseases and cancer. Diseases and conditions in whichactivating a PD1 gene may be useful as a therapeutic treatment includeautoimmune diseases such as systemic lupus erythematosus (SLE).Polynucleotides encoding ZFP TFs or ZFNs may be used as therapeuticsthemselves, or may be incorporated into vectors for delivery.

Methods and compositions comprising ZFP-TFs that repress a PD1 allele,and/or PD1 specific ZFNs may also be used in conjunction with othertherapeutics designed to treat a chronic infectious disease or cancer.These ZFPs or ZFNs (or polynucleotides encoding these ZFPs or ZFNs) maybe administered concurrently (e.g., in the same pharmaceuticalcompositions) or may be administered sequentially in any order. Any typeof cancer can be treated, including, but not limited to lung carcinomas,pancreatic cancers, liver cancers, bone cancers, breast cancers,colorectal cancers, ovarian cancers, leukemias, lymphomas, brain cancersand the like. Similarly, ZFP TFs designed to activate a PD1 allele maybe used with other therapeutics designed to treat an autoimmune disease.

Methods and compositions for treatment also include cell compositionswherein a mutant copy of the PD1 allele within cells isolated from apatient have been modified to a wild-type PD1 allele using aPD1-specific ZFN. These ex vivo modified cells are then reintroducedinto the patient. Additionally, methods and compositions comprisingmodified stem cells are also envisioned. For example, stem cellcompositions wherein a mutant copy of the PD1 allele within the stemcells has been modified to a wildtype PD1 allele using a PD1-specificZFN. In other embodiments, stem cell compositions are provided wherein awild-type PD1 allele within the stem cells has been modified usingPD1-specific ZFNs. These compositions may be used in conjunction withother therapeutics. These compositions may be administered concurrently(e.g., in the same pharmaceutical compositions) or may be administeredsequentially in any order.

The methods and compositions of the invention are also useful for thedesign and implementation of in vitro and in vivo models, for example,animal models of chronic infection, cancer or autoimmunity, which allowsfor the study of these disorders and furthers discovery of usefultherapeutics.

The following Examples relate to exemplary embodiments of the presentdisclosure in which the nuclease comprises a ZFN. It will be appreciatedthat this is for purposes of exemplification only and that othernucleases can be used, for instance homing endonucleases (meganuclases)with engineered DNA-binding domains and/or fusions of naturallyoccurring of engineered homing endonucleases (meganuclases) DNA-bindingdomains and heterologous cleavage domains.

EXAMPLES Example 1 Identification of Persistently Biologically ActivePD1-Specific ZFNs

ZFNs were assembled against the human PD1 gene and were tested by ELISAand CELL assays as described in Miller et al. (2007) Nat. Biotechnol.25:778-785 and U.S. Patent Publication No. 20050064474 and InternationalPatent Publication WO2005/014791

Specific examples of ZFPs are disclosed in Table 1. The first column inthis table is an internal reference name (number) for a ZFP. Table 2lists target binding sites on PD1. “F” refers to the finger and thenumber following “F” refers to which zinc finger (e.g., “F1” refers tofinger 1).

TABLE 1  Human PD1-targeted zinc finger proteins Design SBS # F1 F2 F3F4 F5 F6 12942 QSGHLSR RSDSLSV HNDSRKN RSDDLTR RSDHLTQ N/A (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 1) NO: 2) NO: 3) NO: 4) NO: 5) 12946RSAALSR RSDDLTR RSDHLTT DRSALSR DRSALAR N/A (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 6) NO: 4) NO: 7) NO: 8) NO: 9 ) 12947 RSAALARRSDDLSK RNDHRKN DRSALSR DRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 10) NO: 11) NO: 12) NO: 8) NO: 9) 12934 RSDHLSE TSSDRTKRSDHLSE QSASRKN N/A N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 13) NO: 14)NO: 13) NO: 15) 12971 RSDVLSE RSANLTR RSDHLSQ TSSNRKT DRSNLSR RSDALAR(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 16) NO: 17) NO: 18)NO: 19) NO: 20) NO: 21) 12972 DDWNLSQ RSANLTR RSDHLSQ TSSNRKT DRSNLSRRSDALAR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 22) NO: 17)NO: 18) NO: 19) NO: 20) NO: 21) 18759 RSSALSR RPLALKH RNDHRKN TRPVLKRDRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 23) NO: 24)NO: 12) NO: 25) NO: 9) 22237 QSGHLSR RSDSLSV HNDSRKN RANSLLR RSDHLTQ N/A(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 1) NO: 2) NO: 3) NO: 26)NO: 5) 25005 RPSTLHR RSDELTR RNNNLRT TRPVLKR DRSALAR N/A (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: 27) NO: 28) NO: 29) NO: 25) NO: 9) 25006RPSTLHR RSDELTR TNWHLRT TRPVLKR DRSALAR N/A (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 27) NO: 28) NO: 30) NO: 25) NO: 9) 25010 RPSTLHRRSDELTR RTPHLTL TRPVLKR DRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 27) NO: 28) NO: 31) NO: 25) NO: 9) 25011 RPSTLHR RSDELTRRSAQLAT TRPVLKR DRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: 27) NO: 28) NO: 32) NO: 25) NO: 9) 25012 RPSTLHR RSDELTR RCTHLYLTRPVLKR DRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 27)NO: 28) NO: 33) NO: 25) NO: 9) 25013 RPSTLHR RSDELTR RPTQRYS TRPVLKRDRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 27) NO: 28)NO: 34) NO: 25) NO: 9) 25014 RPSTLHR RSDELTR RANHREC TRPVLKR DRSALAR N/A(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 27) NO: 28) NO: 35) NO: 25)NO: 9) 25015 RPSTLHR RSDELTR RANHREC TRPVLKR DRSALAR N/A (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: 27) NO: 28) NO: 35) NO: 25) NO: 9) 25016RKFARPS RNFSRSD HPHHRMC TRPVLKR DRSALAR N/A (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 36) NO: 37) NO: 38) NO: 25) NO: 9) 25017 RPSTLHRRSDELTR RMGRLST TRPVLKR DRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 27) NO: 28) NO: 39) NO: 25) NO: 9) 25022 RPSTLHR RSDELTRRHSRLTT TRPVLMR DRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: 27) NO: 28) NO: 40) NO: 41) NO: 9) 25023 RPSTLHR RSDELTR RANHRVCTRPVLKR DRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 27)NO: 28) NO: 42) NO: 25) NO: 9) 25025 RPSTLHR RSDELTR RSTHLLG TRPVLKRDRSALAR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID N/A NO: 27) NO: 28)NO: 43) NO: 25) NO: 9) 25027 RNAALTR RSDELTR RSCGLWS TRPVLKR  DRSALAR(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID N/A NO: 45) NO: 28) NO: 44)NO: 25) NO: 9) 25028 CNAALTR RSDELTR REEHRAT TRPVLKR DRSALAR (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID N/A NO: 46) NO: 28) NO: 47) NO: 25)NO: 9) 25029 RNAALTR RSDELTR RHHHLAA TRPVLKR DRSALAR N/A (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: 45) NO: 28) NO: 48) NO: 25) NO: 9) 25030RNAALTR RSDELTR RPMHLTN TRPVLKR DRSALAR N/A (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 45) NO: 28) NO: 49) NO: 25) NO: 9) 25031 RNAALTRRSDELTR RSPHLYH TRPVLKR DRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 45) NO: 28) NO: 50) NO: 25) NO: 9) 25032 RNAALTR RSDELTRRCEALHH TRPVLKR DRSAQAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: 45) NO: 28) NO: 51) NO: 25) NO: 52) 25034 RNAALTR RSDELTR  RCEALHHTRPVLKR DRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 45)NO: 28) NO: 51) NO: 25) NO: 9) 25036 RNAALTR RSDELTR RSPHLYH TRPVLKRDRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 45) NO: 28)NO: 50) NO: 25) NO: 9) 25040 RNAALTR RSDELTR RLPALLS TRPVLKR DRSALAR N/A(SEQ ID (SEQ ID (SEQ ID (SEQ ID  (SEQ ID NO: 45 NO: 28) NO: 53) NO: 25)NO: 9) 25041 HNAALTR RSDELTR RTYNRTQ TRPVLKR DRSALAR N/A (SEQ ID (SEQ ID (SEQ ID  (SEQ ID (SEQ ID NO: 54) NO: 28) NO: 55) NO: 25) NO: 9)

TABLE 2  ZFN Target sites in the human PD1gene SBS# Target site 12942ccAGGGCGCCTGTGGGAtctgcatgcct (SEQ ID NO: 56) 12946caGTCGTCTGGGCGGTGctacaactggg (SEQ ID NO: 57) 12947caGTCGTCTGGGCGGTGctacaactggg (SEQ ID NO: 57) 12934gaACACAGGCACGGctgaggggtcctcc (SEQ ID NO: 58) 12971ctGTGGACTATGGGGAGCTGgatttcca (SEQ ID NO: 59) 12972ctGTGGACTATGGGGAGCTGgatttcca (SEQ ID NO: 59) 18759caGTCGTCTGGGCGGTGct (SEQ ID NO: 60) 22237ccAGGGCGCCTGTGGGAtctgcatgcct (SEQ ID NO: 56) 25005caGTCGTCTGGGCGGTGct (SEQ ID NO: 60) 25006caGTCGTCTGGGCGGTGct (SEQ ID NO: 60) 25010caGTCGTCTGGGCGGTGct (SEQ ID NO: 60) 25011caGTCGTCTGGGCGGTGct (SEQ ID NO: 60) 25012caGTCGTCTGGGCGGTGct (SEQ ID NO: 60) 25013caGTCGTCTGGGCGGTGct (SEQ ID NO: 60) 25014caGTCGTCTGGGCGGTGct (SEQ ID NO: 60) 25015caGTCGTCTGGGCGGTGct (SEQ ID NO: 60) 25016caGTCGTCTGGGCGGTGct (SEQ ID NO: 60) 25017caGTCGTCTGGGCGGTGct (SEQ ID NO: 60) 25022caGTCGTCTGGGCGGTGct (SEQ ID NO: 60) 25023caGTCGTCTGGGCGGTGct (SEQ ID NO: 60) 25025caGTCGTCTGGGCGGTGct (SEQ ID NO: 60) 25027caGTCGTCTGGGCGGTGct (SEQ ID NO: 60) 25028caGTCGTCTGGGCGGTGct (SEQ ID NO: 60) 25029caGTCGTCTGGGCGGTGct (SEQ ID NO: 60) 25030caGTCGTCTGGGCGGTGct (SEQ ID NO: 60) 25031caGTCGTCTGGGCGGTGct (SEQ ID NO: 60) 25032caGTCGTCTGGGCGGTGct (SEQ ID NO: 60) 25034caGTCGTCTGGGCGGTGct (SEQ ID NO: 60) 25036caGTCGTCTGGGCGGTGct (SEQ ID NO: 60) 25040caGTCGTCTGGGCGGTGct (SEQ ID NO: 60) 25041caGTCGTCTGGGCGGTGct (SEQ ID NO: 60)

Initial in vitro activity assays were performed on nucleofected cellsamples as described (above Briefly, the plasmid encoding ZFP-FokIfusions were introduced into K562 cells by transfection using the Amaxa™Nucleofection kit as specified by the manufacturer. For transfection,two million K562 cells were mixed with varying amounts of eachzinc-finger nuclease expression plasmid and 100 μL Amaxa™ Solution V.Cells were transfected in an Amaxa Nucleofector II™ using program T-16.Immediately following transfection, the cells were divided into twodifferent flasks and grown in RPMI medium (Invitrogen) supplemented with10% FBS in 5% CO₂ at either 30° C. or 37° C. for four days.

From this initial in vitro screen, two lead ZFN pairs were identifiedand submitted for elaboration in order to try to improve theirefficiency. These pairs target exons 1 and 5 of the PD1 gene,respectively. The elaborated (improved) proteins were retested in atime-course experiment, essentially as described above. The results aresummarized in Table 3 below.

TABLE 3 PD1 NHEJ % NHEJ Target ZFN pair Day 3 Day 7 Day 9 exon 112942/12946 8 7 5 exon 1 12942/12947 10 6 6 exon 5 12934/12971 11 6 1.5exon 5 12934/12972 11 7.5 2

As shown in Table 3, treatment of cells with ZFNs against exon 5 causesthe loss of a greater proportion of genome-edited cells from thepopulation, while the genome-editing signal in cells treated with ZFNsdesigned against exon 1 is much more stable.

To determine the ZFN activity at the PD1 locus, Cel-1 based SURVEYOR™Nuclease assays were performed essentially as per the manufacturer'sinstructions (Trangenomic SURVEYOR™). Cells were harvested andchromosomal DNA prepared using a Quickextract™ Kit according tomanufacturer's directions (Epicentre®). The appropriate region of thePD1 locus was PCR amplified using Accuprime™ High-fidelity DNApolymerase (Invitrogen). PCR reactions were heated to 94° C., andgradually cooled to room temperature. Approximately 200 ng of theannealed DNA was mixed with 0.33 μL, Cel-I enzyme and incubated for 20minutes at 42° C. Reaction products were analyzed by polyacrylamide gelelectrophoresis in 1×Tris-borate-EDTA buffer.

Constructs were also tested in primary PBMC samples that had been heldat either 30° C. or 37° C. for either 3 or 10 days (see Table 4).Briefly, PBMC were obtained from AllCells and were cultured in RPMI+10%FBS+1% L-Glutamine (30 mg/mL)+IL-2 (1 ng/mL, Sigma) and activated withanti-CD3/CD28 beads according to the manufacturer's protocol (Dynal).Cells were seeded at 3E5 cell/mL in 1 mL volume in a 24 well plate.

Adenoviral vectors were constructed containing the ZFN pairs of interestas described (see US Patent Publication 20080159996) and were added twodays later at an MOI of 10, 30, or 100 (MOI calculated based oninfectious titer).

Cells were harvested 3 or 10 days after exposure to virus and genemodification efficiency was determined using a Cel-I based SURVEYOR™Nuclease assay, performed as described in International PatentPublication WO 07/014,275. See, also, Oleykowski et al. (1998) NucleicAcids res. 26:4597-4602; Qui et al. (2004) BioTechniques 36:702-707;Yeung et al. (2005) BioTechniques 38:749-758.

For the ZFN pairs shown in Table 4, each ZFN was tested in combinationwith ZFN 12942. Activity is measured by percent NHEJ activity asmeasured by the Cel-1 based SURVEYOR™ Nuclease assay described above.

Additional pairs of PD1 specific ZFNs were also tested for activity inprimary PBMC as described above, and the results are shown in Table 4.In the data shown in Table 4, the PD1-specific monomer 12942 was alwayspaired with the second ZFN listed in Table 4 to form an active pair(i.e. ZFN 12942 was paired with each of ZFN 12947 through 25041). See,also, FIG. 1 (samples are as indicated in Table 4).

TABLE 4 Activity of PD1 ZFNs 37° C. 37° C. 30° C. 30° C. Day 3 Day 10Day 3 Day 10 (Percent (Percent (Percent (Percent Sample in 12942+ NHEJ)NHEJ) NHEJ) NHEJ) FIG. 1 12947 2.1 1.6 4.2 2.3 1 18759 4.7 2.2 4.9 4.3 225005 4.4 2.3 4.5 2.4 3 25006 2.9 8.1 5.2 9.9 4 25010 4.9 1.8 5.0 2.8 525011 3.1 9.2 3.1 12.8 6 25012 5.9 8.5 8.2 14.7 7 25013 3.7 0.6 4.0 1.88 25014 10.7 6.6 8.3 9.6 9 25015 3.9 3.9 5.3 7.3 10 25016 7.3 12.8 7.713.6 11 25017 7.7 9.6 6.1 15.0 12 25022 3.6 5.2 4.2 9.0 13 25023 3.1 8.37.1 7.8 14 25025 8.8 10.6 6.5 7.6 15 25027 6.0 9.5 5.9 6.6 16 25028 4.35.2 4.8 6.2 17 25029 8.1 12.8 7.6 14.3 18 25030 7.6 9.6 5.4 10.7 1925031 9.4 14.5 3.8 15.3 20 25032 6.7 4.2 6.6 6.0 21 25034 4.9 4.7 6.24.1 22 25036 8.3 4.2 6.9 9.7 23 25040 6.1 3.6 3.6 4.6 24 25041 7.9 11.25.2 4.5 25

To assay the local effects of the ZFN driven NHEJ activity at themolecular level, CD8+ cells were treated with the exon1 specific ZFNpair 12942 and 12947. Briefly, CD8+ cells were purchased from AllCellsand were cultured in RPMI+10% FBS+1% L-Glutamine (30 mg/mL)+IL-2 (30μg/mL, Sigma) and allowed to rest for 4-24 hours.

Plasmids were constructed containing the ZFN pairs of interest asdescribed above and 1e6 cells/nucleofection were used with the Amaxa™Nucleofection kit as specified by the manufacturer. Cells were activated12-24 hours post nucleofection with anti-CD3/CD28 beads according to themanufacturer's protocol (Dynal).

Cells were harvested 3 or 10 days after nucleofection and genemodification efficiency was determined using a Cel-1 based SURVEYOR™Nuclease assay, performed as described in International PatentPublication WO 07/014,275. See, also, Oleykowski et al. (1998) NucleicAcids res. 26:4597-4602; Qui et al. (2004) BioTechniques 36:702-707;Yeung et al. (2005) BioTechniques 38:749-758.

PCR products were cloned and transfected into E. coli. Antibioticresistant subclones were grown up, the plasmids isolated and thensubjected to sequence analysis to observe any sequence alterations thathad occurred as a result of NHEJ (see FIG. 2). As can be seen from thefigure, a variety of insertions and deletions were observed in thevicinity of the ZFN cleavage site.

These ZFNs were also tested in the yeast system as described in USPublication No. 20090111119.

Example 2 Ex Vivo Activity of PD1-Specific ZFNs in Mice

To test the concept of deleting PD1 in vivo, mouse PD1-specific ZFNswere made as described above and then tested ex vivo. The sequencecharacteristics of the zinc finger domains, as well as their bindingspecificities are shown below in Tables 5 and 6.

TABLE 5  Murine PD1-specific zinc finger designs Design SBS # F1 F2 F3F4 F5 F6 14534 DDWNLSQ RSANLTR TSGSLSR QSGDLTR QSSDLRR N/A (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 22) NO: 17) NO: 61) NO: 62) NO: 63)14534- DDWNLSQ RSANLTR TSGSLSR QSGDLTR QSSDLRR N/A FokI (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID KK NO: 22) NO: 17) NO: 61) NO: 62) NO: 63) 14536QSSHLTR RSDNLRE DRSNLSR TSSNRKT RSDSLSK QSANRTT (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: 64) NO: 65) NO: 20) NO: 19) NO: 66) NO: 80)14536-  QSSHLTR RSDNLRE DRSNLSR TSSNRKT RSDSLSK QSANRTT Fok1 (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID EL NO: 64) NO: 65) NO: 20)NO: 19) NO: 66) NO: 80) 14545 QSGDLTR RSDNLSE ERANRNS DRSDLSR QSSDLRRN/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 62) NO: 67) NO: 68)NO: 69) NO: 63) 14545- QSGDLTR RSDNLSE ERANRNS DRSDLSR QSSDLRR N/A FokI(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID KK NO: 62) NO: 67) NO: 68)NO: 69) NO: 63) 14546 DRSHLAR RSDDLSR QSANRTK RSDTLSE ANSNRIK N/A(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 70) NO: 71) NO: 72) NO: 73)NO: 74) 14546- DRSHLAR RSDDLSR QSANRTK RSDTLSE ANSNRIK N/A FokI (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID EL NO: 70) NO: 71) NO: 72) NO: 73)NO: 74)

TABLE 6 Binding specificities for Murine PD1-specific zinc finger designs SBS#Target site 14534 gtGCTGCAGTTGAGCTGgcaatcagggt (SEQ ID NO: 75)14534-FokI KK gtGCTGCAGTTGAGCTGgcaatcagggt (SEQ ID NO: 75) 14536ccCAAGTGAATGACCAGGGTacctgccg (SEQ ID NO: 76) 14536-FokI ELccCAAGTGAATGACCAGGGTacctgccg (SEQ ID NO: 76) 14545caGCTGCCCAACAGGCAtgacttccaca (SEQ ID NO: 77) 14545-FokI KKcaGCTGCCCAACAGGCAtgacttccaca (SEQ ID NO: 77) 14546atGATCTGGAAGCGGGCatcctggacgg (SEQ ID NO: 78) 14546-FokI ELatGATCTGGAAGCGGGCatcctggacgg (SEQ ID NO: 78)

On day 1, splenocytes harvested from Pmel TCR transgenic/Rag1−/− micewere processed into a single cell suspension and resuspended in completemedia (RPMI-1640, 10% Fetal Bovine Serum, 0.1 mM nonessential aminoacids, 1 mM sodium pyruvate, 2 mM L-glutamine, 50 μg/ml gentamicinsulfate, 50 μM 2-mercaptoethanol) with 25 μA of Invitrogen MouseT-Activator CD3/CD28 Dynabeads/10⁶ cells. Cells were plated in 24 wellplate at 2×10⁶ cells/ml (2 ml/well). On day 3, cells were harvested,separated from beads using a magnet and counted. Transfection ofsplenocytes was performed following the protocol provided with theAmaxa™ Nucleofection kit. Briefly, 1×10e⁷ viable cells were resuspendedin 100 μA of Amaxa Nucleofector Solution and transfected either with 4μg of plasmid containing ZFN pair 14546 and 14545, or 4 μg of mut PD1Fok1 plasmid containing ZFN pair 14546-FokI EL and 14545-Fok I KK, 2.5μg of Amaxa pmax GFP vector, or a no DNA control, using AmaxaNucleofector (program X-01). Cells were cultured in 2 ml of fullysupplemented Amaxa Mouse T Cell Nucleofector Medium at 30° C. aftertransfection. The next day, one ml of Amaxa Mouse T cell NucleofectorMedium was removed and replaced with one ml of complete mediasupplemented with 10 U/ml IL-2. Two days later, cells were harvested andcounted. Two million cells from the each of the groups were plated inanti-CD3 coated wells on a 24 well plate. The next day, cells wereharvested and stained with anti-PD1 PE; anti-CD3 PE-Cy7, and anti-CD8APC. Cells were analyzed on a BD FACSCalibur. Plots show % PD1 positiveCD3+CD8+ cells in each group and the median PD1 fluorescence for eachgroup (see FIG. 3). The data shows that PD1 expression is decreased inthe cells that received the PD1-specific ZFNs, even in the presence ofanti-CD3 stimulation.

To verify that the reduction in PD1 expression was evident at later timepoints, cells were also harvested at 72 hours post anti-CD3 stimulation,rather than at 24 hours as described above. Cells were harvested andstained with anti-PD1 PE; anti-CD3 PE-Cy7, and anti-CD8 APC. Cells wereanalyzed on a BD FACSCalibur. The data is presented in FIG. 4. The upperhistograms show % PD1 positive CD3+CD8+ cells in each group and themedian PD1 fluorescence for each group. Lower plots show the frequencyof PD1/CFSE expressing cells. Importantly, mut PD1 Fok1 and wt PD1 Fok1show higher frequency of PD1^(neg) CFSE^(dim) (dividing cells) thancontrol groups, and demonstrate that PD1 expression is still decreasedin the cells treated with the PD1-specific ZFNs even 72 hours postanti-CD3 stimulation.

Example 3 Activity of Human PD1-Specific ZFNs in TILs

Human PD1-specific ZFNs were tested in human tumor infiltratinglymphocytes (TILs) in the presence of tumors and assayed essentially asdescribed above and using methods known in the art (see for exampleYoshino et al, (1992) Cancer Research 52: 775-781). PD1-specific ZFNswere activated using anti-CD3 antibodies as described above, then thecells were transduced with Ad5/F35 adenovirus expressing PD1-specificZFNs. Cells were expanded with IL2 and then restimulated with anti-CD3antibodies or with tumors and assayed 24 hours post stimulation. Theresults are shown in Table 7 below.

TABLE 7 PD1 expression and viability in TILs CD3 stimulation Tumorextracts 12942 EL/12947 12942 GFP KK 12942/12947 GFP EL/12947 KK12942/12947 PD1 expression 32.2% 31.5% 14.1% 22.9% 13.8% 7.5% %viability TIL 30.3% 34.2% 45.6%   18%   32% 47.9% % viability tumor N/AN/A N/A 45.7% 33.1% 19.6% cell % PD1 + divided  1.6%  0.9%  0.3%  1.1% 0.3% 0.1% cells % PD1 − divided  3.9%  3.0%  2.1%  3.0%  1.4% 0.8%cells

The data in Table 7 demonstrate that when cells are stimulated byanti-CD3 antibodies, decreased PD1 expression, through the action of thePD1 specific ZFNs, leads to increased cell viability. When thetransduced cells are treated with tumors, the same phenomenon isobserved—the ZFN mediated decrease in PD1 leads to an increase in TILviability. Also, the data shows that a decrease in PD1 expression in thetransduced TILs leads to a decrease in tumor cell viability.

Example 4 Purification of PD1 Edited Primary Human T Cells

The PD1 specific ZFN pairs that had been further elaborated were testedin CD4+ T cells as described above in Example 1. As seen in FIG. 5, upto 44% editing was observed with some pairs. In this experiment,‘Positive Control’ indicates cutting using the 25025/12942 ZFN pair inCD8+ T cells performed previously under different experimentalconditions.

One lead pair, 25029/12942 was chosen for further use in isolating PD1modified cells. Briefly, in these experiments, CD4+ T cells were treatedwith mRNA encoding the PD1 specific ZFNs, cultured under the “30 degree”conditions and then stimulated with a first exposure to anti-CD3/CD8beads (Dynal) as described above in Example 1, which stimulates strongexpression of the ZFN transgenes and promotes cleavage at the PD1 locus(see United States Patent Publication No. 20080311095). Following thisfirst stimulation, the cells were re-stimulated and subjected to apurification procedure, either by FACs or by affinity chromatography.

Briefly, the CD4+ T cells were treated either with CCR5-specific ZFNs(see United States Patent Publication 20080159996) or the PD1-specificZFNs. Cells were collected and analyzed for PD1 editing by the Cel-Iassay (described above), i) following the first stimulation, ii)following the second stimulation but prior to any purification, iii)following cell sorting for CD25+ (a marker of activation), PD1(−) usingstandard methodology, or iv) after affinity chromatography using amatrix made with either anti-PD1 antibody or anti-CD25 antibody.

As shown in FIG. 6, using the cell sorting technique (where cells wereisolated that were positive for CD25 but negative for PD1), up to 56% ofthe recovered cells were found to be modified as assayed by the Cel-1assay. PD1(−) cells purified by the affinity chromatography technique(where cells were subjected to affinity matrices made using eitheranti-PD1 antibodies, anti-CD25 antibodies, or both) displayed an overallPC1 modification of up to 42% as assayed by Cel-1 analysis.

Cells that had been purified by cell sorting were also analyzed at theirPD1 locus by sequencing, and the results are presented below in Table 8.As can be seen from the table, the percent target modification (‘%NHEJ’) predicted by the Cel-I analysis is similar to that found by thesequencing analysis. In this table, the ‘Sample’ label corresponds tothose shown in FIG. 6.

TABLE 8 Percent PD1 modification in CD25+ cells % NHEJ by % NHEJ byNumber Sample Cel I sequencing modified* insertions deletions 4 43 62 54of 87 3 of 54 51 of 54 8 56 81 65 of 80 4 of 65 61 of 65 13 42 59 43 of73 1 of 43 42 of 43 *‘Number modified’ indicates the number of sequencesin the sequencing group that were observed to be modified. For example,in sample 4, 54 sequences of the 87 analyzed were modified.

The PD1 specific ZFNs were also tested in CD8+ T cells. In thisexperiment, mRNAs encoding the PD1 specific ZFNs were produced using theRibomax Large Scale RNA ProductionT7 kit (Promega), followed by theRNeasy mini kit (Qiagen), both according to the manufacturer'sprotocols. Varying amounts of mRNAs were used to transduce the cellsusing the Amaxa Nucleofection delivery system as described above, andthe percent PD1 modification was analyzed by the Cel I assay.

As shown in FIG. 7, the amount of modification observed, as described as‘% NHEJ’, is related to the amount of mRNA used, with lesser amounts ofinput mRNA resulting in lesser percentages of target modification.

These results demonstrate that the PD1 specific ZFNs described hereinare capable of modifying the PD1 locus in cell lines and in primary Tcells.

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entirety.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity of understanding,it will be apparent to those skilled in the art that various changes andmodifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing descriptions andexamples should not be construed as limiting.

What is claimed is:
 1. A method of modifying PD1 expression in a cell ofa subject having a disease or disorder characterized by aberrantexpression or regulation of a PD1 gene, the method comprising modulatingexpression of the PD1 in the cell of the subject by administering apolynucleotide encoding a fusion protein comprising a zinc fingerprotein and a functional domain to the cell, wherein the zinc fingerprotein comprising four, five or six zinc finger recognition regionsordered from F1 to F4, F1 to F5 or F1 to F6, from N-terminus toC-terminus, and wherein the recognition regions comprise the followingamino acid sequences: (i)  (SEQ ID NO: 1) F1: QSGHLSR, (SEQ ID NO: 2)F2: RSDSLSV, (SEQ ID NO: 3) F3: HNDSRKN, (SEQ ID NO: 4) F4: RSDDLTR, and(SEQ ID NO: 5) F5: RSDHLTQ; (ii) (SEQ ID NO: 6) F1: RSAALSR,(SEQ ID NO: 4) F2: RSDDLTR, (SEQ ID NO: 7) F3: RSDHLTT, (SEQ ID NO: 8)F4: DRSALSR, and (SEQ ID NO: 9) F5: DRSALAR; (iii) (SEQ ID NO: 10)F1: RSAALAR, (SEQ ID NO: 11) F2: RSDDLSK, (SEQ ID NO: 12) F3: RNDHRKN,(SEQ ID NO: 8) F4: DRSALSR, and (SEQ ID NO: 9) F5: DRSALAR; (iv)(SEQ ID NO: 13) F1: RSDHLSE, (SEQ ID NO: 14) F2: TSSDRTK,(SEQ ID NO: 13) F3: RSDHLSE, and (SEQ ID NO: 15) F4: QSASRKN; (v)(SEQ ID NO: 16) F1: RSDVLSE, (SEQ ID NO: 17) F2: RSANLTR,(SEQ ID NO: 18) F3: RSDHLSQ, (SEQ ID NO: 19) F4: TSSNRKT,(SEQ ID NO: 20) F5: DRSNLSR, and (SEQ ID NO: 21) F6: RSDALAR; or (vi)(SEQ ID NO: 22) F1: DDWNLSQ, (SEQ ID NO: 17) F2: RSANLTR,(SEQ ID NO: 18) F3: RSDHLSQ, (SEQ ID NO: 19) F4: TSSNRKT,(SEQ ID NO: 20) F5: DRSNLSR, and (SEQ ID NO: 21) F6: RSDALAR; (vii)(SEQ ID NO: 23) F1: RSSALSR, (SEQ ID NO: 24) F2: RPLALKH,(SEQ ID NO: 12) F3: RNDHRKN, (SEQ ID NO: 25) F4: TRPVLKR, and(SEQ ID NO: 9) F5: DRSALAR; (viii) (SEQ ID NO: 1) F1: QSGHLSR,(SEQ ID NO: 2) F2: RSDSLSV, (SEQ ID NO: 3) F3: HNDSRKN, (SEQ ID NO: 26)F4: RANSLLR, and  (SEQ ID NO: 5) F5: RSDHLTQ; (ix)  (SEQ ID NO: 27)F1: RPSTLHR, (SEQ ID NO: 28) F2: RSDELTR, (SEQ ID NO: 29) F3: RNNNLRT,(SEQ ID NO: 25) F4: TRPVLKR, and (SEQ ID NO: 9) F5: DRSALAR; (x)(SEQ ID NO: 27) F1: RPSTLHR, (SEQ ID NO: 28) F2: RSDELTR, (SEQ ID NO: 30) F3: TNWHLRT, (SEQ ID NO: 25) F4: TRPVLKR, and(SEQ ID NO: 9) F5: DRSALAR; (xi) (SEQ ID NO: 27) F1: RPSTLHR,(SEQ ID NO: 28) F2: RSDELTR, (SEQ ID NO: 31) F3: RTPHLTL,(SEQ ID NO: 25) F4: TRPVLKR, and (SEQ ID NO: 9) F5: DRSALAR; (xii)(SEQ ID NO: 27) F1: RPSTLHR, (SEQ ID NO: 28) F2: RSDELTR,(SEQ ID NO: 32) F3: RSAQLAT, (SEQ ID NO: 25) F4: TRPVLKR, and(SEQ ID NO: 9) F5: DRSALAR; (xiii) (SEQ ID NO: 27) F1: RPSTLHR,(SEQ ID NO: 28) F2: RSDELTR, (SEQ ID NO: 33) F3: RCTHLYL,(SEQ ID NO: 25) F4: TRPVLKR, and (SEQ ID NO: 9) F5: DRSALAR; (xiv)(SEQ ID NO: 27) F1: RPSTLHR, (SEQ ID NO: 28) F2: RSDELTR,(SEQ ID NO: 34) F3: RPTQRYS, (SEQ ID NO: 25) F4: TRPVLKR, and (SEQ ID NO: 9) F5: DRSALAR; (xiv) (SEQ ID NO: 27) F1: RPSTLHR, (SEQ ID NO: 28) F2: RSDELTR,  (SEQ ID NO: 35) F3: RANHREC,(SEQ ID NO: 25) F4: TRPVLKR, and (SEQ ID NO: 9) F5: DRSALAR; (xvi)(SEQ ID NO: 36) F1: RKFARPS, (SEQ ID NO: 37) F2: RNFSRSD,(SEQ ID NO: 38) F3: HPHHRMC, (SEQ ID NO: 25) F4: TRPVLKR, and(SEQ ID NO: 9) F5: DRSALAR; (xvii) (SEQ ID NO: 27) F1: RPSTLHR,(SEQ ID NO: 28) F2: RSDELTR, (SEQ ID NO: 39) F3: RMGRLST,(SEQ ID NO: 25) F4: TRPVLKR, and (SEQ ID NO: 9) F5: DRSALAR; (xviii)(SEQ ID NO: 27) F1: RPSTLHR, (SEQ ID NO: 28) F2: RSDELTR,(SEQ ID NO: 40) F3: RHSRLTT, (SEQ ID NO: 41) F4: TRPVLMR, and(SEQ ID NO: 9) F5: DRSALAR; (xix) (SEQ ID NO: 27) F1: RPSTLHR,(SEQ ID NO: 28) F2: RSDELTR, (SEQ ID NO: 42) F3: RANHRVC,(SEQ ID NO: 25) F4: TRPVLKR, and  (SEQ ID NO: 9) F5: DRSALAR; (xx)(SEQ ID NO: 27) F1: RPSTLHR, (SEQ ID NO: 28) F2: RSDELTR, (SEQ ID NO: 43) F3: RSTHLLG,  (SEQ ID NO: 25) F4: TRPVLKR, and (SEQ ID NO: 9) F5: DRSALAR; (xxi)  (SEQ ID NO: 45) F1: RNAALTR,(SEQ ID NO: 28) F2: RSDELTR (SEQ ID NO: 44) F3: RSCGLWS (SEQ ID NO: 25)F4: TRPVLKR, and (SEQ ID NO: 9) F5: DRSALAR; (xxii) (SEQ ID NO: 46)F1: CNAALTR, (SEQ ID NO: 28) F2: RSDELTR, (SEQ ID NO: 47) F3: REEHRAT,(SEQ ID NO: 25) F4: TRPVLKR, and  (SEQ ID NO: 9) F5: DRSALAR; (xxiii)(SEQ ID NO: 45) F1: RNAALTR, (SEQ ID NO: 28) F2: RSDELTR,(SEQ ID NO: 48) F3: RHHHLAA, (SEQ ID NO: 25) F4: TRPVLKR, and(SEQ ID NO: 9) F5: DRSALAR; (xxiv) (SEQ ID NO: 45) F1: RNAALTR,(SEQ ID NO: 28) F2: RSDELTR, (SEQ ID NO: 49) F3: RPMHLTN,(SEQ ID NO: 25) F4: TRPVLKR, and (SEQ ID NO: 9) F5: DRSALAR; (xxv)(SEQ ID NO: 45) F1: RNAALTR, (SEQ ID NO: 28) F2: RSDELTR,(SEQ ID NO: 50) F3: RSPHLYH, (SEQ ID NO: 25) F4: TRPVLKR, and (SEQ ID NO: 9) F5: DRSALAR; (xxvi) (SEQ ID NO: 45) F1: RNAALTR,(SEQ ID NO: 28) F2: RSDELTR, (SEQ ID NO: 51) F3: RCEALHH,(SEQ ID NO: 25) F4: TRPVLKR, and (SEQ ID NO: 52) F5: DRSAQAR; (xxvii)(SEQ ID NO: 45) F1: RNAALTR, (SEQ ID NO: 28) F2: RSDELTR,(SEQ ID NO: 51) F3: RCEALHH, (SEQ ID NO: 25) F4: TRPVLKR, and (SEQ ID NO: 9) F5: DRSALAR; (xxviii) (SEQ ID NO: 45) F1: RNAALTR,(SEQ ID NO: 28) F2: RSDELTR, (SEQ ID NO: 53) F3: RLPALLS,(SEQ ID NO: 25) F4: TRPVLKR, and  (SEQ ID NO: 9) F5: DRSALAR; and (xxix)(SEQ ID NO: 54) F1: HNAALTR, (SEQ ID NO: 28) F2: RSDELTR,(SEQ ID NO: 55) F3: RTYNRTQ, (SEQ ID NO: 25) F4: TRPVLKR, and(SEQ ID NO: 9) F5: DRSALAR.


2. The method of claim 1, wherein expression of the PD1 gene isrepressed.
 3. The method of claim 1, wherein the fusion proteincomprises a cleavage domain and the PD1 gene is inactivated.
 4. Themethod of claim 1, wherein the disease or disorder is a cancer, a viralinfection or an autoimmune disease.
 5. The method of claim 1, furthercomprising administering additional anti-viral or anti-cancer therapiesto the subject.
 6. The method of claim 5, wherein the additionalanti-viral or anti-cancer therapies are administered prior to,concurrently or sequentially with the polynucleotide encoding the fusionprotein.
 7. The method of claim 1, wherein the cell is modified ex vivoand administered to the subject following modification.
 8. The method ofclaim 1, wherein the cell is a tumor infiltrating lymphocyte (TIL). 9.The method of claim 1, wherein the cell is a CD4+ T-cell.